Arrestins

CHAPTER FOUR

Arrestins: Role in the Desensitization, Sequestration, and Vesicular Trafficking of G Protein-Coupled Receptors Cornelia Walther*,†, Stephen S.G. Ferguson*,†

*J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute, Western University Canada, London, Ontario, Canada † Department of Physiology and Pharmacology, Western University Canada, London, Ontario, Canada

Contents 1. Introduction 2. Arrestins in GPCR Desensitization 3. Arrestins in GPCR Trafficking 3.1 Sequestration 3.2 Postendocytic vesicular trafficking of GPCRs 4. Conclusions Acknowledgments References

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Abstract Over the years, b-arrestins have emerged as multifunctional molecular scaffolding proteins regulating almost every imaginable G protein-coupled receptor (GPCR) function. Originally discovered as GPCR-desensitizing molecules, they have been shown to also serve as important regulators of GPCR signaling, sequestration, and vesicular trafficking. This broad functional role implicates b-arrestins as key regulatory proteins for cellular function. Hence, this chapter summarizes the current understanding of the b-arrestin family’s unique ability to control the kinetics as well as the extent of GPCR activity at the level of desensitization, sequestration, and subsequent intracellular trafficking.

1. INTRODUCTION G protein-coupled receptors (GPCRs) are integral membrane proteins that represent the largest and functionally most diverse family of cell-surface receptor proteins. Members of this family share a common Progress in Molecular Biology and Translational Science, Volume 118 ISSN 1877-1173 http://dx.doi.org/10.1016/B978-0-12-394440-5.00004-8

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2013 Elsevier Inc. All rights reserved.

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topographical organization with seven transmembrane spanning a-helices connected by three intracellular and three extracellular loops with extracellular amino and intracellular carboxyl termini. GPCRs transduce the information provided by a multitude of physical and chemical extracellular stimuli into intracellular second messengers and thereby have the ability to mediate and modulate essential biological functions. However, besides their numerous important roles in basic cellular physiology, dysregulation of GPCR function eventually results in pathophysiologic conditions. As a consequence, understanding the molecular mechanisms underlying GPCR function is fundamental to the development of new drugs for a host of human diseases. The classical paradigm for transduction of external signals across the plasma membrane into the cell following ligand binding to GPCRs involves the coupling of GPCRs to heterotrimeric guanine nucleotide-binding proteins (G proteins), which promotes the exchange of GDP for GTP on the G protein a subunit. This, in turn, leads to the dissociation of Ga and Gbg subunits.1 The activated subunits subsequently regulate the activity of a wide variety of effectors, thus regulating intracellular second messenger levels that mediate the cellular response to receptor activation. Besides the G protein-dependent activation of downstream effectors such as ion channels, phospholipases, and adenylyl cyclases, agonist-mediated GPCR activation also results in multiple molecular protein interactions that initiate (1) receptor desensitization, (2) receptor endocytosis, (3) intracellular trafficking between intracellular vesicular compartments, (4) the activation of G protein-independent signaling pathways, and (5) either receptor resensitization or downregulation.2 These cellular processes display remarkable kinetic differences. Whereas desensitization occurs within seconds, endocytosis takes place over minutes and resensitization ensues within minutes to hours.3 All of these processes are governed by a myriad of intracellular accessory proteins, generally termed GPCR-interacting proteins (GIPs). Within the last two decades of research, the list of GIPs has expanded rapidly and continues to grow.4–8 Notably, one of the first GIPs identified is the cytosolic protein arrestin, which was first shown to bind to GRK1-phosphorylated rhodopsin.9 The arrestin family in vertebrates is now known to consist of four members: two visual arrestins, arrestin-1 and arrestin-4, that are limited in their expression to the phototransduction pathway (retinal rods and cones), and the two nonvisual arrestins, b-arrestin-1 and b-arrestin-2 (alternatively known as arrestin-2 and arrestin-3). b-Arrestin-1 and b-arrestin-2 are ubiquitously expressed

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in mammalian tissue and are known to contribute to the uncoupling of many GPCRs from heterotrimeric G proteins.10,11 For many years, b-arrestin actions were thought to be limited to the desensitization and internalization processes of GPCRs.12–15 However, over the past several years, b-arrestins have emerged as one of the key players involved in the regulation of multiple facets of GPCR function; hence, they have evolved as multifunctional cellular mediators.16 Besides the well-known participation of b-arrestins in receptor desensitization, due to their ability to uncouple GPCR/G protein complexes, b-arrestins have also been identified as signal scaffolding proteins as they contain specific interaction sites for various signaling as well as accessory molecules.12,16,17 Consequently, b-arrestins not only have the capacity to regulate GPCR desensitization, they also control a vast array of additional cellular functions including cell signaling as well as membrane, cytosolic, and nuclear-associated trafficking of GPCRs.16 Intense research has been conducted to identify the kinetics of b-arrestin binding, as well as the molecular determinants that dictate whether b-arrestins bind to a given GPCR and/or subsequently regulate GPCR activity at multiple steps within the GPCR life cycle. This chapter will focus on the current understanding of how and to what extent b-arrestins control GPCR desensitization, sequestration, and vesicular trafficking.

2. ARRESTINS IN GPCR DESENSITIZATION Receptor desensitization represents an important physiological process that prevents GPCRs from overstimulation due to prolonged agonist exposure by signal attenuation or termination.2,3 The classical model of GPCR desensitization involves three processes: (1) receptor phosphorylation and subsequent uncoupling of the receptor from its cognate G protein, (2) receptor sequestration (internalization) to intracellular compartments, and (3) downregulation. Desensitization is typically elicited by three classes of regulatory molecules: (1) second messenger-dependent protein kinases, (2) G protein-coupled receptor kinases (GRKs), and (3) b-arrestins.3,11 Second messenger-dependent protein kinases, such as protein kinase A and C, that mediate heterologous desensitization, phosphorylate GPCRs at specific consensus sequences within intracellular loops or carboxyl terminal domains that prevent G protein coupling. However, second messenger-dependent protein kinases do not discriminate between inactive and agonist-activated GPCRs. Hence, they prevent the activation of GPCRs that have never been exposed to agonists as well as receptors that

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have been agonist activated. There is no evidence that second messengerdependent protein kinase phosphorylation-dependent uncoupling of GPCRs from heterotrimeric G proteins requires the association of b-arrestin with GPCRs.3 In contrast, phosphorylation by GRKs (homologous desensitization) occurs only following the agonist-dependent isomerization of GPCRs to an activated state that results in GRK-mediated phosphorylation of distinct serine and threonine residues within intracellular GPCR domains.18 Specifically, GRKs phosphorylate agonist-activated GPCRs because the activated receptor itself activates the kinase.19 The GRK family consists of seven members (GRK1–7) that display different distribution patterns and show receptor-specific preferences.20–22 Whereas GRK1 and 7 expression is restricted to retinal rods and cones and GRK4 displays limited expression in the kidneys, testis, and cerebellum, GRK2, -3, -5, and -6 are ubiquitously expressed in mammalian tissues.22,23 However, GRKmediated phosphorylation is not sufficient to cause desensitization of most GPCRs, but rather requires the recruitment of the cytosolic cofactor protein arrestin. b-Arrestins bind to agonist-activated and phosphorylated GPCRs and serve to sterically uncouple receptors from heterotrimeric G proteins resulting in the termination of G protein-dependent signal transduction10,11,24 (Fig. 4.1). Accordingly, the first function of b-arrestins is to

Figure 4.1 Classical model of G protein-coupled receptor kinase (GRK)-mediated G protein-coupled receptor (GPCR) desensitization. Agonist binding leads to conformational changes allowing for coupling and activation of heterotrimeric G proteins. This leads to the specific phosphorylation of agonist-activated receptors by GRKs at various intracellular domains, primarily intracellular loop three and the carboxyl terminus. Subsequently, arrestin is recruited to the phosphorylated receptor, where it sterically uncouples the receptor from its cognate G protein and, in turn, leads to receptor desensitization. A, agonist; G, G protein; GRK, G protein-coupled receptor kinase; P, phosphate moiety; b-ARR, b-arrestin.

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“arrest” receptor signaling via G proteins. However, there is clearly a tight interplay of GRKs and b-arrestins to control the desensitization process. It has been found for numerous receptors that predominantly GRK2 and GRK3 over GRK5 and GRK6 are responsible for phosphorylation and subsequent b-arrestin recruitment.25,26 However, several studies exist, where primarily GRK5 and GRK6 have been implicated in desensitization, for example, calcitonin gene-related peptide receptors27 and dopamine D1A receptors.28 A proposed GRK-specific “bar code”29,30 dictates subsequent downstream effects and whether or not a specific b-arrestin isoform binds to the GPCR. For example, GRK2- and GRK3-mediated phosphorylation of CXCR4 receptors leads to b-arrestin-1 recruitment, whereas GRK2-/6dependent events involve b-arrestin-2 binding.31 Comparably, the stimulation of the CCR7 receptor by different ligands either leads to the activation of both GRK3 and GRK6 or just GRK6 alone. Different phosphorylation patterns subsequently lead to distinct b-arrestin recruitment: either to the membrane or to endocytic vesicles.32 Thus, it has become evident that the cellular GRK repertoire, depending on the respective receptor and tissue studied, may dictate the characteristics and scope of receptor responsiveness and desensitization.28 Generally, b-arrestin binding to GRK-phosphorylated receptors is determined by the absence or presence of conserved clusters of Ser/Thr residues within the carboxyl terminal tails of the receptor. GPCRs can be broadly divided into two classes, A and B, depending on the stability of b-arrestin binding to the receptor.33,34 The carboxyl terminal tails of class A receptors, such as the b2-adrenergic receptor (b2AR), consist of a diffusely Ser/Thr-rich cluster and only transiently recruit b-arrestin-2 upon phosphorylation. In contrast, class B receptors like the V2 vasopressin receptor and the angiotensin II type 1A receptor recruit b-arrestin-1 as well as b-arrestin-2 with high affinity and allow for the stable association of b-arrestin with the receptors. How is this b-arrestin selectivity mechanistically achieved? A “multisite arrestin-receptor interaction” model has been proposed to account for the differences.35 This model hypothesizes that b-arrestins have two binding sites: one that binds distinct receptor elements which are subject to conformational change upon receptor activation, and a second one that binds receptor-attached phosphates. When a GPCR is activated and subsequently phosphorylated and b-arrestin binds via both interaction sites, it is subject to transition into its active high-affinity receptor-binding state. This mechanism most likely contributes to the preferential desensitization of certain

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GPCRs by b-arrestins.22 To bind arrestins with high affinity, many GPCRs must be phosphorylated. This was first shown for rhodopsin36 and later for numerous other GPCRs such as the b2AR,24,37–40 angiotensin II type 1 receptor,41 a2-adrenergic receptor,42 m2 muscarinic receptor,38 and neuropeptide Y1 receptor.43 The majority of relevant phosphorylation sites are localized to the carboxyl terminal tails of most GPCRs, but relevant phosphorylation sites have been mapped to any intracellular domain including the intracellular loops (as reviewed in Ref. 44). Nonetheless, b-arrestins were also found to bind to several unphosphorylated receptors.45–47 Phosphorylation-independent b-arrestin binding can be explained by acidic amino acid stretches within intracellular domains. Acidic amino acids can successfully mimic phosphate groups48,49; thus, the density of negative charges is sufficient to activate the arrestin phospho-binding site which leads to receptor interaction, such has been demonstrated for the D6 chemokine receptor.50 Due to the vast complexity of phosphorylation events that occur on different GPCRs, it is still a matter of debate how only seven GRKs and four arrestins can specifically regulate several hundred different GPCRs. For many GPCRs, it appears that all GRKs or arrestins (except retinal arrestins) contribute to the regulation of their desensitization. However, many GPCRs display a distinct preference for a specific GRK and arrestin isoform to mediate their desensitization. For example, GRK6 and b-arrestin-2 regulate m-opioid receptors and D2-like dopamine receptors in striatum.21 It has also become evident that the regulation of a given GPCR depends on cell background, as its regulation can be affected by differences in the expression levels of distinct GRKs and b-arrestins between different cell types and tissues. It remains an open question whether certain GPCRs are regulated either randomly by various GRK/b-arrestin combinations or by specific GRK and b-arrestin pairs, and whether a particular GRK/b-arrestin pairing takes precedence over other pairings.21 To add an additional level of complexity to GRK and b-arrestin regulation of GPCR activity, Schulz et al.51 recently identified a novel mechanism whereby distinct opioid agonists stimulated site-specific m-opioid receptor phosphorylation patterns. Thus, morphine binding results in a m-opioid receptor conformation with next to no affinity for b-arrestins, whereas enkephalin analogs induce a rapid high-affinity binding of both b-arrestin-1 and -2.51,52 Similarly, the chemokine receptor CCR2 displays different affinities for both b-arrestin-1 and -2, depending on whether the receptor was stimulated with different CCR2 ligands. Furthermore, while CCL7 induces transient binding of b-arrestins, CCL8/13 leads to the stable formation

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of a CCLR7/b-arrestin complex. Consequently, stimulation with different ligands stabilizes different receptor conformations, leading to qualitative differences in arrestin responses.53 The importance of arrestins in GPCR desensitization has been clearly demonstrated in studies using Drosophila and mice. Drosophila strains that display mutations in the arrestin gene show impaired inactivation of metarhodopsin.54 Similarly, b-arrestin-2 knock-out mice exhibit a significant potentiation and prolongation of morphine-induced analgesia indicating altered m-opioid receptor desensitization.55 In addition, and apart from its well-established and extensively studied function in uncoupling the GPCR from its cognate G protein, more recent studies discovered novel roles for arrestins in the regulation of GPCR desensitization. b-Arrestins have been identified to serve as shuttle proteins that localize phosphodiesterases within the vicinity of agonist-activated bARs.56 In cardiac myocytes, b-arrestinmediated phosphodiesterase recruitment to activated bARs promotes the switch from Gs to Gi coupling and subsequently results in bAR signaling shifting toward a pathway that limits cAMP production.57 In addition, b-arrestins have been shown to reduce the level of the second messenger diacylglycerol upon agonist stimulation of the M1 cholinergic receptor.58 These results suggest novel impacts of b-arrestins on GPCR desensitization, as they presumably function to limit the generation of second messengers, but are also capable of enhancing the rate of second messenger degradation.16 These findings clearly expand the functional relevance of arrestin in GPCR desensitization.

3. ARRESTINS IN GPCR TRAFFICKING While desensitization serves to protect receptors from overstimulation, receptor sequestration (internalization) is required to prevent prolonged desensitization, as well as to enable GPCRs to either resensitize or become downregulated upon agonist removal.2,3 In addition to the well-established importance of b-arrestins in desensitization, they also play a central role in mediating GPCR endocytosis. b-Arrestins function as endocytic adaptor proteins, recruiting membrane-bound activated receptors to the internalization machinery.59 The process of agonist-induced sequestration leads to the functional removal of activated receptors from the plasma membrane and targets them to intracellular compartments for either cell surface recycling and resensitization or degradation. But the question arises as to how this is accomplished mechanistically. It turns out that the underlying

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mechanism(s) for b-arrestin-dependent regulation of GPCR trafficking is far more complex than was initially assumed. This is because b-arrestins display varying binding affinities to different GPCRs depending on the cell type studied.

3.1. Sequestration The sequestration of GPCRs is essential to the maintenance and regulation of agonist responsiveness, as it can result in not only dephosphorylation, recycling, and resensitization of the receptor but also in receptor degradation and activation of G protein-independent intracellular signaling pathways.22 The first evidence that b-arrestins have functions beyond arresting G protein-mediated signaling was reported in 1996.60 In this pioneering study, endocytosis-deficient b2AR mutants were rescued by the overexpression of wild-type b-arrestins and b2AR internalization was prevented by the expression of dominant-negative b-arrestin mutants. The sequestration of most GPCRs requires GRK phosphorylation and b-arrestin binding such that these events are indispensible prerequisites. However, depending on the GRK involved in receptor phosphorylation and subsequent b-arrestin binding, distinct downstream processes are favored. For example, it has been shown that only GRK2-mediated phosphorylation initiates b-arrestin-dependent endocytosis of the V2 vasopressin and angiotensin II type 1A receptors.25,26 In contrast, vasopressin and angiotensin receptor phosphorylation by GRK5 and GRK6 triggers b-arrestin-dependent activation of ERK pathways, rather than internalization. Similar effects have been described for b2AR, as well as for the follicle-stimulating hormone receptor.25,26,31 In general, three common mechanisms have been implicated in GPCR internalization. These mechanisms either involve (1) clathrin-coated pits, (2) caveolae, or (3) other yet to be characterized uncoated vesicles.61,62 Whereas GRK-mediated phosphorylation and b-arrestin binding are critical to clathrin-dependent sequestration,14 receptors that lack GRK phosphorylation sites are sequestered via caveolae in a b-arrestin-independent manner, for example, the b1AR.63 b-Arrestins function as adaptor molecules to link GPCRs to the clathrin-coated pit machinery through their ability to bind directly and stoichiometrically to the major structural components of the endocytic machinery.64–66 Such components include the heavy chain of clathrin,67 as well as b-adaptin, a component of the clathrin-associated adaptor protein complex AP-2.68,69 Clathrin was discovered as the first

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nonreceptor b-arrestin-interacting protein in 1996.67 AP-2 is involved in the recruitment of clathrin and the subsequent assembly of clathrin lattices, the major components of the coat of the internalized membrane. Binding of clathrin and AP-2 is facilitated by b-arrestin binding to the receptor, which leads to the release of the b-arrestin carboxy-terminal tail, the region where both interaction partners bind. Thus, clathrin and AP-2 binding to b-arrestins during endocytosis has turned out to be a critical step in internalization.12 Whether GPCR sequestration involves clathrin-coated pits, caveolae, and/or other uncoated vesicles, internalization rates are highly receptor and cell-type dependent. Almost every possible internalization mechanism for different GPCRs has been described in the literature: b-arrestin- and clathrin-dependent, b-arrestin- and clathrin-independent, b-arrestinindependent and clathrin-dependent, and also b-arrestin-dependent and clathrin-independent.62 Thus, it seems that b-arrestins are sometimes redundant for the internalization of some GPCRs.70 There is also evidence that certain receptors can choose different internalization pathways and can also dictate whether they internalize via either b-arrestin-dependent or independent pathways. For example, the chemokine receptor CCR5 and m2 muscarinic receptors internalize in a b-arrestin-dependent manner and, although bound to b-arrestin, can also internalize in a b-arrestin-independent manner.71 Other examples include the full length A2B adenosine receptor and the neuropeptide Y2 receptor, which typically internalize via a b-arrestindependent pathway. However, carboxyl terminal truncations of both receptors serve to redirect them to a b-arrestin-independent internalization pathway.72,73 Thus, b-arrestins provide GPCRs with the ability to interact with the clathrin-coated pit internalization machinery, but this interaction does not necessarily predetermine whether GPCRs are endocytosed in clathrin-coated vesicles. However, it is now clear that the majority of GPCRs utilize the b-arrestin-dependent, clathrin-mediated internalization pathway (Fig. 4.2). The two nonvisual mammalian b-arrestins display different characteristics with respect to cellular distribution patterns, and binding properties to their GPCR substrates. While b-arrestin-1, comparable to visual arrestin, is localized in the cytoplasm and the nucleus, b-arrestin-2 is limited to the cytoplasm.34,74 They also have different abilities to mediate internalization. Evidence to support this has come from studies with knockout mice either lacking b-arrestin-1 or -2. Although both b-arrestin-1 and -2 contribute to the desensitization of the b2AR, b-arrestin-2 appears to be significantly

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Figure 4.2 The prototypical model of b-arrestin-promoted sequestration of GPCRs via the clathrin-mediated internalization pathway. Agonist-activated phosphorylated receptors are guided to clathrin-coated pits by the recruitment of b-arrestin, which binds to AP-2 and clathrin. The vesicles are subsequently pinched off by the GTPase dynamin, which leads to receptor internalization into early endosomes.

more efficient at promoting its internalization.75 In contrast, activation of the angiotensin II type 1 receptor results in the recruitment of both b-arrestin-1 and -2, which display comparable efficiency in desensitizing and internalizing the receptor.34,75 These findings have led to the classification of GPCRs into two subgroups that reflect the stability and outcome of GPCR/arrestin interactions. Class A receptors, including b2AR, m-opioid receptors, endothelin type A receptors, and dopamine D1A receptors, have a higher affinity for b-arrestin-2 and form transient GPCR/arrestin complexes that dissociate at/near the cell surface. In contrast, class B receptors, such as V2 vasopressin, neurotensin 1, and angiotensin II type 1A receptors, bind b-arrestin-1 and -2 with equivalent affinity and form stable GPCR/b-arrestin complexes that remain intact as the receptors endocytose.34,76,77 Chimeric receptors (b2AR/angiotensin II type 1 receptors) have provided strong evidence that the stability of GPCR/b-arrestin complexes are controlled by determinants within the GPCR carboxyl terminal tail domains. Consequently, we now know that the receptor

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carboxyl terminus determines the stability of the GPCR/b-arrestin complex and subsequent cellular distribution of b-arrestin.76 This is likely achieved by the distinct phosphorylation patterns induced by different GRKs, the density of phosphorylation sites within a given GPCR C-tail or intracellular loop domain, and the induction of conformational changes in receptor-bound b-arrestins, as these conformational states may vary and will likely reflect dynamic changes in the binding of downstream accessory proteins.33,44 The list of GPCRs that have been shown to bind to b-arrestins via the phosphorylated Ser/Thr residues within their carboxyl terminal tails initially consisted only of the rhodopsin receptor and the b2AR. However, due to intense investigation in this field over the past two decades worldwide, this list is continuously expanding. To date, evidence for receptor/b-arrestin interactions involved in internalization is provided for b2AR,60,78,79 M2muscarinic acetylcholine receptors,70,80,81 d-opioid receptors,82 angiotensin II type 1 receptors,83 human P2Y1 receptors,84 dopamine D1 receptors,85 neuropeptide Y1, Y2, and Y4 receptors,43,73,86,87 and many more. The contribution of specific intracellular receptor domains to b-arrestin binding and subsequent sequestration has been studied intensively. Most commonly, the carboxyl terminus (prototypical model) and, to a lesser extent, the third intracellular loop contribute to endocytic trafficking. However, even within the class of adrenergic receptor subtypes, specific differences occur. For example, b-arrestins bind to the carboxyl terminus of the b2AR, but they bind to the third intracellular loop of a-adrenergic receptors.88 Similar characteristics have been documented for the muscarinic acetylcholine receptors 2 and 3.88,89 Clearly, receptor domains are significantly involved in the regulation of sequestration. Another important regulator of b-arrestin-promoted clathrin-dependent receptor internalizations is the posttranslational modification of b-arrestin itself. b-Arrestin-1 is regulated by phosphorylation/dephosphorylation. Ser412 within the carboxyl termini of b-arrestin-1 is constitutively phosphorylated and recruited to the plasma membrane as soon as the receptor is activated in an agonist-dependent manner. Subsequently, b-arrestin-1 becomes dephosphorylated, which is required for clathrin binding and targeting to clathrin-coated pits.49 Besides phosphorylation, ubiquitination of b-arrestins is another required process for effective internalization. b-Arrestin-2 binds the mouse double minute 2 (mdm2) protein, an E3 ubiquitin ligase, and becomes ubiquitinated, a process shown to regulate the stability of the b2AR/b-arrestin interaction internalization.90 Likewise,

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b-arrestin ubiquitinylation is fundamental to subsequent clathrin-mediated endocytosis.15 Recently, a new interaction partner for b-arrestin, phosphatidylinositol 4-phosphate 5-kinase, has been shown to be involved in the agonistmediated sequestration of the b2AR receptor. The complex formation of b-arrestin-2, phosphatidylinositol 4-phosphate 5-kinase, and the agonistbound b2AR appears to be essential to regulate receptor internalization.91 Thus, strong evidence is provided that the function of b-arrestins at the level of GPCR sequestration goes far beyond the fact that b-arrestin binds agonist-activated and GRK-phosphorylated receptors.

3.2. Postendocytic vesicular trafficking of GPCRs Once GPCRs are sequestered and transported into intracellular compartments, b-arrestins also have the ability to influence the intracellular fate of GPCRs by regulating their postendocytic trafficking. The majority of GPCRs are either recycled or degradated. It is still largely unknown how cells decide which pathway a particular receptor will take. There is a growing body of evidence suggesting that cells decide on the ultimate fate of certain receptors based on the properties of the receptor/b-arrestin complex. The division of GPCRs into class A and B reflects the apparent affinities for b-arrestin. While class A receptors, which show higher affinity for b-arrestin-2 than b-arrestin-1, traffic to endosomes and recycle directly back afterward, class B receptors, which bind both b-arrestins equally well, form a stable complex with arrestin and are either retained in endosomes for hours before being recycled or are targeted to lysosomes for final degradation.20,33,34,76,92 Apparently, b-arrestin and its receptor affinity represent a critical determinant for GPCR postendocytic sorting as it determines the rate of receptor resensitization (Fig. 4.3). Evidence is provided by studies with chimeric b2AR and V2 vasopressin receptors: exchange of the carboxyl terminal tails of the rapidly recycling b2AR and the extremely slow recycling V2 vasopressin receptor completely reverses the pattern of b-arrestin binding and the propensity of these receptors to dephosphorylate, recycle, and resensitize.92 The same effect was observed for the exchange of the C-termini of two vasopressin receptor subtypes: V1A, which recycles rapidly, and V2, which moves slowly. Exchange of the carboxyl termini between these two receptors also switches the trafficking pattern of the receptors.93 As the stable association of b-arrestin is promoted by a specific cluster of phosphorylated Ser/Thr residues within the carboxyl terminus, the interaction of b-arrestins with such a specific phospho-cluster, a

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Figure 4.3 Postendocytic trafficking of GPCRs strongly depends on the stability of receptor/arrestin complexes as well as the interaction with various other intracellular proteins that interact with the receptor-bound b-arrestin during the internalization process. The magnified region depicts those arrestin-interacting proteins influencing internalization and thus subsequent trafficking to intracellular compartments. As internalization is the prerequisite for subcellular trafficking, these arrestin-interacting proteins are involved in both mechanisms and boundaries are blurred. Once internalized, GPCRs can be divided into two classes, depending on the properties of the receptor–arrestin complex. Class A receptors, which preferentially bind b-arrestin-2, dissociate rapidly from the bound arrestin, become dephosphorylated, and transit through recycling endosomes before reaching the plasma membrane for efficient resensitization. In contrast, class B receptors, which recruit both arrestins with equal affinity, remain associated with arrestin during internalization and are targeted to lysosomes for final degradation. Occasionally, class B receptors are retained in early endosomes for a prolonged period before undergoing recycling. A, agonist; P, phosphate moiety; b-ARR, b-arrestin; ARF6, G protein ADP-ribosylation factor 6; ARNO, ARF nucleotidebinding site opener; Mdm2, mouse double minute protein 2, an E3 ubiquitin ligase; NSF, N-ethylmaleimide-sensitive fusion protein; Ub, Ubiquitin.

predetermined distance from the seventh transmembrane domain, appears to dictate the rate of dephosphorylation, recycling, and resensitization.92 Studies examining b2AR endocytosis in the presence of b-arrestin mutants that have either decreased or increased affinities for GPCRs and endocytic adaptor proteins revealed that arrestin directs the receptor to recycling or degradation pathways, respectively.94,95 Therefore, it can be concluded that

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b-arrestins regulate a GPCR’s ability to resensitize and ultimately reestablish responsiveness.96 Both b-arrestin expression levels and the rate of b-arrestin release dictate fate.44,96 Not surprisingly, exceptions to the rule exist as not all GPCRs fit into the class A/B dichotomy. Somatostatin (sst) receptors, for example, do not follow the classical postendocytic trafficking patterns for class A and B receptors as described earlier. While the sst2A somatostatin receptor exhibits a typical class B receptor b-arrestin-dependent trafficking pattern (stable b-arrestin–receptor complexes, cointernalization), this receptor is rapidly recycled and resensitized rather than targeted for degradation. In contrast, the somatostatin receptor subtypes sst3 and sst5 display typical class A receptor properties (transient b-arrestin–receptor complexes, receptor internalization without b-arrestin), but unlike other class A receptors, are targeted to lysosomes for degradation.97 Similar observations were made for the spontaneous internalization of the a1A-adrenergic receptor. Although this receptor internalizes in a b-arrestin-dependent manner, it travels with b-arrestin bound to recycling endosomes.98 A third example is the metabotropic glutamate receptor 1, which interacts selectively with b-arrestin1, not b-arrestin-2.99 Even so, for most GPCRs, the stability of the receptor-b-arrestin complex strongly affects the postendocytic trafficking pattern, and thereby determines the ultimate subcellular fate. In the past few years, it has become evident that the capacity of b-arrestins to engage in protein–protein interactions is not limited to receptors and components of the clathrin-mediated endocytosis machinery, or dedicated to the control of receptor desensitization and internalization. The number of identified b-arrestin-interacting partners is growing rapidly. b-Arrestins are capable of forming an interface between intracellular components of the vesicular trafficking and sorting machinery and internalized receptors. Recently, novel intracellular interaction partners involved in arrestin-promoted clathrin-mediated trafficking have been identified. Among them are the small G protein ADP-ribosylation factor 6 and its guanine nucleotide exchange factor ADP-ribosylation factor nucleotidebinding site opener,100 and the N-ethylmaleimide-sensitive fusion protein101 (Fig. 4.3). These proteins subsequently act as adaptors to facilitate the clathrin-mediated endocytosis of GPCRs. Another regulatory factor in b-arrestin-mediated postendocytic sorting is the ubiquitination status of b-arrestin, which dramatically influences the subcellular fate of GPCRs. For example, a constitutively ubiquitinated b-arrestin-ubiquitin chimera is unable to dissociate from the b2AR, and

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its expression in cells directs the receptor toward degradative pathways rather than the naturally occurring recycling and resensitization.15

4. CONCLUSIONS Arrestin function in GPCR desensitization, sequestration, and vesicular trafficking has been studied intensively over decades. Thereby, researchers worldwide were able to establish a prototypical mechanism on how arrestin regulates each of these steps individually. Nonetheless, exceptions to the predominant rules governing the regulation of GPCRs will always exist and may provide advantages for the development of new therapeutics to treat disease. It is increasingly becoming evident that GPCRs are not the only substrates for b-arrestin. The b-arrestin interaction network is far more complex than assumed 20 years ago. Arrestins are now recognized as key scaffolding molecules that connect membrane receptors to multiple cytosolic proteins that use the b-arrestin-bound receptor as the platform for assembling multiple protein signaling complexes. Thus, b-arrestins have the remarkable ability both to regulate the kinetics and the extent of GPCR cellular activity at the level of desensitization, internalization, and subsequent intracellular fate, and control the activation and compartmentation of a wide variety of G protein-independent signal transduction cascades.

ACKNOWLEDGMENTS This work was supported by an operating grant to S. S. G. F. from the Canadian Institutes of Health Research (CIHR) (MOP-119437, MOP-62738, and MOP-111093) and a Navigator Grant from the Huntington’s Society of Canada. S. S. G. F. also holds a Tier I Canada Research Chair in Molecular Neurobiology and is a Career Investigator of the Heart and Stroke Foundation of Ontario.

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